Dirty template DNA

The quality of the template DNA you use in sequencing is absolutely crucial. We can't stress how important it is to provide a template as clean as possible especially for automated sequencing. Any contaminants like salt, protein, carbohydrate, primers, or excess dNTP's will affect your data and there is no way of getting around this. If you look at the example image the peaks for the main data set are OK, but below them are a number of peaks that should not be associated with this data, on the whole this looks at lot "dirtier" than an optimal result as shown in figure 1 on the overview page.

Figure: Dirty Template DNA

You can take care of a number of things to solve this problem. The most important is to have a good and convenient, but reliable, method for DNA purification in the first place and once you find a method that gives good results stick with it. There are a number of commercially available kits for prepping your template DNA and on the whole these are very good for producing automated sequence quality DNA. They either rely on a silica membrane designed for size exclusion ("spin preps", elution of DNA with water) or work as anion exchange columns ("gravity flow" products, elution with high salt). From our experience the use of phenol-chloroform prepared DNA gives very poor results. We have never seen it work in automated sequencing. We use the Millipore 96-well plate format kits as with our own MWG Biotech Robosmart DNA prepping robots they provide consistently high quality DNA. However, all silica membrane based systems give a good template quality little or no no difference between these commercially available kits (e.g. Promega Wizard, Qiagen SpinPrep, Macherey & Nagel Nucleospin, ...) . Anion exchange columns also provide high quality DNA for automated sequencing if you take extra care of the high salt concentration from the elution buffer. Depending on the respective elution buffer provided by the manufacturer we find the template quality to vary slightly. We prefer systems using K-acetate buffers for elution (Genomed's JetStat, Macherey & Nagel's Nucleobond), but others also work well (e.g. Qiagen's Tip20-500 systems). One problem that we regularly encounter is that some people try to save a bit of money and constantly swap prep kits depending who is selling them cheaper at that time, and these people are invariably those that have the greatest problem in producing high quality DNA consistently. Sometimes it is worth spending a little extra money to make sure you get a product that will work. Another cautionary note is to always follow the manufacturers instructions - if they say only use 5ml of LB broth then use this - if you use TB or enhance the culture volume you will overload the column and you will get poor quality DNA from your prep. On the other hand if you use less then you will recover less DNA at the end.